bioenergetics of the mitochondrion: structure & function of atp … · 2020. 2. 12. · what...

MRC Mitochondrial Biology Unit

SCIENTIFIC METHOD & EXPERIMENTAL DESIGN

Bioenergetics of the Mitochondrion:

Structure & Function of ATP Synthase

Max Perutz 1914-2002

“In Science truth always wins”

Boris Ephrussi 1901-1979

Discovered cytoplasmic inheritance

“We cannot discover the truth of an hypothesis by counting the number of people who believe it.”

The Scientific Method: version 1

• Hypothesis

• Experiment to test hypothesis

• Result; accept, reject or modify hypothesis

• Conclusion

The Scientific Method v2: accretion of information

• Collect accurate observations

• Gradually build up a detailed picture

• “Until it becomes unreasonable to hold an opposing view” A. Klug

• Test features experimentally (and by calculation and modelling)

Then Now

Peter Mitchell (1920-1992)

Metabolism in mitochondria

Subunit compositions of F-ATPases

Eubacteria and chloroplasts Mitochondria

The differences provide opportunities for drug discovery

ATP synthase rotor ring sizes 8-17

stator F1 highly conserved

peripheral stalk highly variable

All ATP synthase conserve central features

ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites. Montgomery, M. G., Gahura, O., Leslie, A. G. W., Zikova, E. & Walker, J. E. (2018). Proc. Natl. Acad. Sci. U. S. A. 115, 2102-2107.

Fundamental features of F-ATPases are highly conserved

What do we still need to know about the F-ATPase?

• 1. How is rotation generated during ATP hydrolysis?

• 2. How is rotation generated during ATP synthesis?

• 3. How is the enzyme regulated?

• 4. How is the enzyme assembled?

• 5. Is the permeability transition pore associated?

1. How rotation is generated from ATP hydrolysis

• “Single molecule” rotational data mainly from bacteria

• Structural data almost entirely from mitochondria

• Reconciliation of these two data sets

Kinosita, Yoshida, Noji & colleagues

Structural definition of phosphate release stepp

IF1 stops the enzyme at the catalytic dwell

Thiophosphate stops F1-ATPase at the phosphate release dwell

Active mammalian IF1 is an antiparallel

α-helical dimer in crystals

130 Å

pH regulates oligomeric state and inhibitory activity of IF1

Inactive tetramer converted to active dimer by decreasing pH from 8.0 to 6.5

Cabezon, Butler, Runswick & Walker, J Biol Chem (2000) 275, 25460-25464

pH and Ca2+ regulate the oligomeric state and inhibitory activity of IF1

Boreikaite, Wicky, Watt, Clarke & Walker, PNAS (2019) 116,10354-10359

• Inhibits ATP hydrolysis, but not synthesis

• Inhibitory region intrinsically disordered when free, structured when bound

• Inhibitory activity regulated by pH and [Ca2+]

• Inhibitory activity not regulated by phosphorylation of Ser-14

Properties of IF1

Bason, Montgomery, Leslie & Walker 2015 PNAS 112, 6009-6014

Thiophosphate inhibited bovine F1-ATPase

2. How is rotation driven by the pmf?

• mechanical coupling mechanism

• the proton pathway

W. Junge

H+/ATP = ring symmetry/ 3

Morales et al, 2015 Vinothkumar et al, 2016 Zhou et al, 2015

3. Assembly of human ATP synthase

ATP synthase is assembled from modules

• ATPase evolved in a modular fashion

• ATPase made of three structural modules

• Achieved by different routes in bacteria, yeast and mammals

Walker, J. E. & Cozens, A. L. (1986). Evolution of ATP synthase. Chemica Scripta 26B, 263-272.

Modular evolution of ATP synthase

Modular assembly of yeast ATP synthase

A Tzagoloff and colleagues

Studies of assembly of human ATP synthase

• Remove each subunit selectively by CRISPR-cas9

• Characterise vestigial ATPase by SDS-PAGE, NGE and SILAC

• Examine effect on growth of clonal cells

δ εα α

ATP8 f6.8P

δ εα α

ATP8 f

Δb (or OSCP)

Δe (or g)

Δ6.8kDΔDAPIT

Monomer Monomer Monomer

Monomer

Monomer/dimerDimerOligomer

Complete

δ εα α

ATP8 f

Effect of removal of yeast subunit g on mitochondrial morphology

Control mitochondria Δg mitochondria

Paumard, … & Velours, EMBO J (2002) 21, 221-230

Harner et al (2016), ELife 5e18853

MICOS, mitochondrial inner membrane complex for maintenance of cristae junctions

The mitochondrial

permeability transition pore

Mitochondrial permeability transition pore

• 1976, Hunter et al

• Allows molecules < 1.5 kDa to pass; diameter 2 nm

• Consequences: mitochondrial depolarization, uncoupling of ox-phos, mitochondrial swelling

• Proposed components: VDAC, ANT

• Inducers: Ca2+, adenine nucleotides, phosphate, oxidative stress

• Inhibitor: Cyclosporin A

Defining features of the PTP

• Opened by elevation of matrix Ca2+

• Opening inhibited by cyclosporin A binding to cyclophilin D

• Opening leads to entry of water and swelling of mitochondria

• Pore estimated to be about 2-2.9 nm in diameter

Induction of the PTP

• Treat cells with thapsigargin

• Permeabilize cytoplasmic membrane with the ionophore ferutinin and introduce exogenous Ca2+

• Permeabilize cells with digitonin and introduce pulses of Ca2+

Pore opening and cyclophilin D

• peptidylprolyl cis-trans isomerase

• mitochondrial version of a larger family

• modulates PTP, but is not a component

• binding of cyclosporin A inhibits pore opening

The Scientific Method

• Observation: mitochondria have a permeability transition pore

• Hypothesis: dimeric ATP synthase or components provide the PTP

• Experiment: systematically remove dimeric ATPase; test the pore

• Result: the pore survives removal of dimeric ATPase

• Conclusion: the dimeric ATPase does not provide the pore

• See: Walker et al (2020) www.pnas.org/cgi/doi/10.1073/pnas.1921409117

Topic 1: The membrane rotors of ATP synthases in various species have a range of symmetries from 8-17. For example for humans it is 8; for Synechococcus it is 15. What are the bioenergetic consequences, and why do you think that the enzymes have evolved in this way?

• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16.

• Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. W. & Walker, J. E. (2010). Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc. Natl. Acad. Sci. U. S. A. 107, 16823-16827.

• Walpole, T. B., Palmer, D. N., Jiang, H., Ding. S., Fearnley, I. M., & Walker, J.E. (2015). Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan ATP synthases. Mol. Cell. Proteomics. 14, 828-840.

Topic 2. In the past 20 years or so, bedaquiline is the only new drug introduced into clinical practice for treatment of tuberculosis. It inhibits the mycobacterial ATP synthase, but how? Given the urgent need for developing new drugs against multiply or even totally resistant bacterial pathogens, what features of the ATP synthase make it an attractive target for development of new drugs against bacterial pathogens, and what are the draw-backs? Remember, ideally the drug should not affect the human enzyme

• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16

• Bason, J., Montgomery, M. G., Leslie A. G. W., & Walker, J. E. (2014). Pathway of binding of the intrinsically disordered mitochondrial inhibitor protein to F1-ATPase. Proc. Natl. Acad. Sci. U. S. A. 11, 11305-11310.

• Andries et al. (2005). A diarylquinoline drug active on the ATP synthase from Mycobacterium tuberculosis. Science, 307, 223-227.

• Preiss et al (2015). Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci. Adv. 1:e1500106

• Zhang et al (2019). The structure of the catalytic domain the ATP synthase from Mycobacterium smegmatis is a target fro developing antitubercular drugs. Proc. Natl. Acad. Sci. U. S. A. 116, 4206-4211.

Topic 3. The permeability transition in human mitochondria refers to the opening of a non-specific membrane channel, known as the permeability transition pore (PTP) in the inner membrane. Opening is triggered by calcium ions, or reactive oxygen species, among other effectors, leading to swelling of the organelle, disruption of the inner membrane, loss of ATP synthesis and cell death. It is an uncharacterized part of cell death by necrosis and possibly apoptosis. Past proposals that the pore is provided by the voltage dependent ion channel in the outer membrane plus the adenine nucleotide translocase in the inner membrane have not withstood scrutiny. A recent proposal is that the pore is associated with the dimeric ATP synthase. How would you test this hypothesis? Remember, you will probably need to assay the functional PTP in a cellular context.

• Dimers of mitochondrial ATP synthase form the permeability transition pore. Georgia et al. (2013) PNAS 110, 5887-5892.

• An uncoupling channel within the c-subunit of the ATP synthase is the permeability transition pore Alavian et al. (2014) PNAS 111, 10580-10585.

Topic 1: The membrane rotors of ATP synthases in various species have a range of symmetries from 8-17. For example for humans it is 8; for Synechococcus it is 15. What are the bioenergetic consequences, and why do you think that the enzymes have evolved in this way?

• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16.

• Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. W. & Walker, J. E. (2010). Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc. Natl. Acad. Sci. U. S. A. 107, 16823-16827.

• Walpole, T. B., Palmer, D. N., Jiang, H., Ding. S., Fearnley, I. M., & Walker, J.E. (2015). Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan ATP synthases. Mol. Cell. Proteomics. 14, 828-840.

c-ring symmetries and H+:ATP ratios

• 8-fold symmetry in metazoans

• 10-fold symmetry in yeast

• 10-15-fold in eubacteria

• 14-fold in chloroplasts

• Therefore, H+/ATP = 2.7-5.0, depending on species

P/O ratio in mammalian mitochondria

e- donor P/O calc P/O exp

NADH 10/3.7 = 2.7 2.5

Succinate 6/3.7 = 1.6 1.5

NADH 2e- to O2 = 10 H+ pumped out Succinate 2e- to O2 = 6 H+ pumped out ADP/ATP exchange + Pi/H+ symport = 1 H+ in

ATP synthesis H+/ATP = 8/3 = 2.7ATP synthesis H+/ATP = 3.7

Moles ADP phosphorylated/2e- to O2 = P/O ratio

Topic 2. In the past 20 years or so, bedaquiline is the only new drug introduced into clinical practice for treatment of tuberculosis. It inhibits the mycobacterial ATP synthase, but how? Given the urgent need for developing new drugs against multiply or even totally resistant bacterial pathogens, what features of the ATP synthase make it an attractive target for development of new drugs against bacterial pathogens, and what are the draw-backs? Remember, ideally the drug should not affect the human enzyme

• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16

• Bason, J., Montgomery, M. G., Leslie A. G. W., & Walker, J. E. (2014). Pathway of binding of the intrinsically disordered mitochondrial inhibitor protein to F1-ATPase. Proc. Natl. Acad. Sci. U. S. A. 11, 11305-11310.

• Andries et al. (2005). A diarylquinoline drug active on the ATP synthase from Mycobacterium tuberculosis. Science, 307, 223-227.

• Preiss et al (2015). Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci. Adv. 1:e1500106

• Zhang et al (2019). The structure of the catalytic domain the ATP synthase from Mycobacterium smegmatis is a target fro developing antitubercular drugs. Proc. Natl. Acad. Sci. U. S. A. 116, 4206-4211.

Inhibition of Staph. aureus by penicillin exuded from the mould

Fleming’s experiment in 1928

Chronology of penicillin development

• 1928 Fleming’s observation

• 1939 Florey, Chain start investigation of penicillin

• 1940 Abraham: penicillin resistant Staph aureus

• 1943 Penicillin used in clinical practice

Nobel Prize in Physiology or Medicine 1945“for the discovery of penicillin and its curative effect in various

infectious diseases”

Emergence of antibiotic resistance

Annual death by antimicrobial resistance (AMR)

Tetanus60000

Cholera110000

Measles130000

AMR700000

Diabetes1500000

Cancers8200000

Diarrhea1300000

Car traffic accidents1300000

AMR in 205010000000

WHO. Review on Antimicrobial Resistance. 2014

Annual deaths from AMR by 2050

Spread of antibiotic resistance

In the USA, animals consume twice as many medically important antibiotics as humans

1940 1950 1960 1970 1980 1990 2000 2010

Sulfa drugsPenicillin

TetracyclineMacroglidesAminoglycosidesChloramphenicol

Discovery gap

Antibiotic era Increasing drug resistance

GlycopeptidesStreptomycinsQuinolinesCephalosporins

OxazolidoninesMutilinsLipopeptides

Tuberculosis

!Disease of the respiratory system; can spread to brain, kidneys and bones

!Highly contagious via coughing

! Symptoms are chest pain, fatigue, weight loss, fever

! 2 billion people infected; 1.6 million estimated deaths in 2017

! 60% untreated die

Why is there a resurgence of TB ?

!Poor adherence to TB control programs

!Emergence of drug resistance - MDR, XRD, TDR

!Widespread synergy with HIV

!No quick and accurate diagnostics

!Outdated drugs and vaccines

Dormancy Resuscitation

MMP, matrix metallo-protease

Control of TB requires

• New drugs against novel targets

• New vaccines - BCG (Bacillus Calmette-Guerin) was introduced in 1921

• Early diagnosis

Shortens anti-tuberculosis treatment (8 weeks) and effective (bactericidal-sterilizing) in patients with drug-susceptible or drug-resistant TB

Most significant discovery in TB research – last 50 years

2 weeks

2-4 months6-30 months

Bedaquiline (SirturoTM)

• First approved anti-TB drug with novel action in 40 years

• Inhibits ATP synthase by binding to the rotor

• Highly specific for mycobacteria

• Active against both dormant and replicating states

• Active against MDR and XDR mycobacteria

Bedaquiline is not a wonder drug

• FDA approval based on phase 2 trials (440 people)

• recommended only for MDR-, XDR- and TDR-TB

• cardiovascular risk

• very lipophilic; human half-life 5-6 months

• patients susceptible to phospholipidosis

• can cause death (10% unexplained)

• need more inhibitors of mycobacterial ATPase

Zhang, Montgomery, Leslie, Cook & Walker, PNAS (2019) 116, 4206-4211

The catalytic domain of ATP synthase from M. smegmatis as a drug target

Topic 3. The permeability transition in human mitochondria refers to the opening of a non-specific membrane channel, known as the permeability transition pore (PTP) in the inner membrane. Opening is triggered by calcium ions, or reactive oxygen species, among other effectors, leading to swelling of the organelle, disruption of the inner membrane, loss of ATP synthesis and cell death. It is an uncharacterized part of cell death by necrosis and possibly apoptosis. Past proposals that the pore is provided by the voltage dependent ion channel in the outer membrane plus the adenine nucleotide translocase in the inner membrane have not withstood scrutiny. A recent proposal is that the pore is associated with the dimeric ATP synthase. How would you test this hypothesis? Remember, you will probably need to assay the functional PTP in a cellular context.

• Dimers of mitochondrial ATP synthase form the permeability transition pore. Georgia et al. (2013) PNAS 110, 5887-5892.

• An uncoupling channel within the c-subunit of the ATP synthase is the permeability transition pore Alavian et al. (2014) PNAS 111, 10580-10585.

Human HAP1 cells

• near haploid human cell line used with CRISPR-cas9 • derived from myeloid leukaemia • fragment of chr15 in chr19, and reciprocal translocation chr 9 and 22• ATP5G1-G3 on chrs 17, 12 and 2

ATP5G1 & ATP5G2 Dyer & Walker, Biochem J (1993) 293, 51-64 ATP5G3 Yan et al (1994) Genomics 24, 375-377

Expression of subunit c in human HAP1cells

Identification of a HAP1 clone devoid of subunit c

J He, H Ford, J Carrol, S Ding, I M Fearnley & J E Walker, PNAS (2017) 114, 3409-3414

Mitochondrial F-ATPase and the PTP: conclusions

• subunit c is not involved in forming the PTP

• nor are subunits ATP6 and ATP8

Ca2+ retention capacity in permeabilised HAP1-WT, Δb and ΔOSCP cells

HAP1-WT

HAP1-Δb

HAP1-ΔOSCP

-CsA +CsA

Summary of findings

• Removal of subunit c and [ATP6 + ATP8] has no effect on the pore

• Removal of e, f, g, 6.8PL or DAPIT has no effect on the pore

• Removal of b or OSCP has no effect on the pore

• In δ[c + δ] cells, there is no ATP synthase, but still a PTP

• ….. pore size is same as in WT-cells

• ….. inhibition by cyclosporin A, BKA and CATR as in WT-cells

• ATP SYNTHASE DOES NOT PROVIDE THE PTP

bioenergetics of the mitochondrion: structure & function of atp … · 2020. 2. 12. · what...

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